Japan Advanced Institute of Science and Technology
Title プレローディング薬物のための金型不使用マイクロニードル
の作製
Author(s) PITAKJAKPIPOP, HARIT Citation
Issue Date 2022-03
Type Thesis or Dissertation Text version ETD
URL http://hdl.handle.net/10119/17776 Rights
Description Supervisor:松村 和明, 先端科学技術研究科, 博士
Mold-less microneedle fabrication for pre-loading drug
Harit Pitakjakpipop
Supervisor: Professor Kazuaki Matsumura
Graduate School of Advanced Science and Technology Japan Advanced Institute of Science and Technology
Materials Science
March 2022
Referee-in-chief:
Professor Dr. Kazuaki Matsumura
Japan Advanced Institute of Science and Technology
Referees:
Professor Dr. Yuzuru Takamura
Japan Advanced Institute of Science and Technology Professor Dr. Toshiaki Taniike
Japan Advanced Institute of Science and Technology Associate Professor Dr. Eijiro Miyako
Japan Advanced Institute of Science and Technology Associate Professor Pakorn Opaprakasit
Sirindhorn International Institute of Technology,
Thammasat University
Abstract
The microneedle technology gets high attention in transdermal drug delivery systems (TDDS) research due to the limitations in the oral and parenteral drug delivery systems. Herein, the photolithography microneedle fabrication method, mold-less, has been employed to overcome the limitation of other fabrication methods. It takes less than 5 minutes for each fabrication process that includes adjusting the length and shape of microneedle by varying the time for UV irradiation and the pattern micro-windows on the photomask that is suitable to produce for industrial scale. Four-point star-shaped microneedles are fabricated via a photolithography process, and sulfobetaine (SPB) monomer is combined with dextran-glycidyl methacrylate/acrylic acid (Dex-GMA/AAc) to form the hydrogel network. The toxicity study shows that the Dex-GMA/AAc/SPB polymer can be considered as extremely biocompatible.
The microneedle itself exhibits high drug loading capacity, high-efficiency drug release, and inhibits protein aggregation. The acrylated epoxidized soybean oil (AESO) sheet, microneedle substrate, shows a clear and flexible property and non-absorb drug during the drug loading process; when the microneedle patch is applied on the skin, it also curves along the surface that demonstrating its ability to be easy to apply to any body part. A pre-drug loading platform is designed for the advanced features of the microneedles that provide an effective option for administering therapeutic drugs.
The rhodamine B drug loading and releasing models show that the microneedle can load drug up to 8µg on one microneedle patch and release up to 80% of loading in 6h, with only 41 needles. The microneedle has the potential for chemical drug delivery due to its propensity for greater loading and releasing efficiency in microneedle applications. The Dextran-FITC diffusion represented that the molecular drug weight lower than 10 kDa can absorb/diffuse into the center of the microneedle. The Dextran-FITC showed a fast release and diffusion into the artificial skin in a short period.
The protein delivery shows some limitation of protein loading and releasing at low pH, but at pH 7.4, protein releases show higher efficiency suitable for transdermal drug delivery, owing to the presence of pH 7.4 in the interstitial fluid. The Lactate dehydrogenase (LDH) enzyme activity study illustrates that the poly-SPB side chains in the Dex-GMA/SPB/AAc hydrogel inhibit protein aggregation while loading enzyme into the microneedle according to having a higher enzyme kinetic activity than Dex-GMA/AAc hydrogel that do not have the poly-SPB side chains even under external stress, releases the proteins in their native state (without activity loss). A ThT assay determining the fibril formation in human insulin indicates that the human insulin-loaded Dex-GMA/AAc and Dex-GMA/SPB/AAc effectively suppress the formation of fibrils in human insulin under the dry condition and high-temperature condition that presented only 25% and 20% aggregation, respectively. The combination of hydrogel microneedle and poly-SPB side chains has a potential for biopharmaceutical transdermal drug delivery, especially protein base drug that increases the efficiency bioavailability.
Keywords: microneedles, photolithography, transdermal drug delivery, zwitterionic polymers, protein aggregation
Contents
Chapter 1 ... 1
1.1 Transdermal drug delivery ... 1
1.1.1 Skin layer... 2
1.2 Microneedle ... 3
1.2.1 Microneedle technology ... 3
1.2.2 Microneedle materials ... 4
1.2.2.1 Metal microneedle ... 4
1.2.2.2 Silicon microneedle ... 5
1.2.2.3 Polymeric microneedle ... 6
1.3 Fabrication methods ... 7
1.4 Drug delivery... 19
1.5 Hydrogel ... 20
1.5.1 Hydrogel in drug delivery ... 20
1.5.2 Dextran ... 21
1.6 Smart microneedle... 23
1.7 Objective of study ... 24
1.8 References ... 26
Chapter 2: Polymer synthesis and mold-less microneedle fabrication ... 33
2.1 Introduction ... 33
2.1.1 Microneedle Classifications ... 34
2.2 Materials and Methods ... 41
2.2.1 Materials ... 41
2.2.2 Synthesis of AESO resin ... 41
2.2.3 Synthesis of Dex-GMA ... 42
2.2.4 Synthesis of Dex-GMA/AAc ... 42
2.2.5 Mold-less Microneedle Fabrication ... 43
2.3 Results and discussion ... 49
2.3.1 AESO Substrate and Surface Characterization ... 49
2.3.1 Characterization of the Dextran-GMA ... 51
2.3.3 Photomask ... 52
2.3.4 Microneedle fabrication ... 53
2.3.5 Drug loading capacity ... 56
2.4 Conclusion ... 59
2.5 References ... 60
Chapter 3 Protein aggregation inhibition microneedle for transdermal drug delivery 64 3.1 Introduction ... 64
3.1.1 Protein aggregation for transdermal drug delivery ... 64
3.1.2 Zwitterionic polymers ... 64
3.2 Materials and methods ... 68
3.2.1 Materials ... 68
3.2.2 Synthesis of Dex-GMA ... 68
3.2.3 Synthesis of Dex-GMA/SPB/AAc resin ... 69
3.2.4 Synthesis of Dex-GMA/AAc resin and PEG-DA resin ... 69
3.2.5 Synthesis of AESO resin ... 70
3.2.6 Preparation of microneedle patches ... 70
3.2.7 Swelling study ... 71
3.2.8 Microneedle mechanical property test ... 72
3.2.9 Drug loading studies... 72
3.2.10 Drug diffusion and release studies ... 73
3.2.11 Protein loading... 74
3.2.12 Protein Release Study... 75
3.2.13 Bradford protein assay ... 75
3.2.14 Determination of protein protection within the microneedle materials . 75 3.2.15 Fibril Formation of Human Insulin ... 76
3.2.16 Parafilm penetration profile ... 77
3.2.17 Porcine skin insertion ... 77
3.2.18 Cytotoxic assay ... 78
3.3 Results and discussion ... 79
3.3.1 Synthesis and Characterization of Dex-GMA/AAc and Dex- GMA/SPB/AAc ... 79
3.3.2 Microneedle Fabrication Process ... 80
3.3.3 Swelling Study ... 82
3.3.4 Microneedle Mechanical Properties ... 83
3.3.5 Drug Loading Capacity ... 86
3.3.6 Drug Release ... 87
3.3.7 Drug Diffusion Study ... 88
3.3.8 Skin Penetration of Microneedle Patches... 92
3.3.9 Protein Loading ... 93
3.3.10 Protein Release ... 93
3.3.11 Dex-GMA/SPB/AAc Microneedles Avoid Protein Denaturation ... 96
3.3.12 Cytotoxicity Study... 99
3.4 Conclusion ... 101
3.5 References ... 103
Chapter 4: General Conclusion ... 111
Achievements ... 115
Acknowledgment ... 116
List of Figures
Figure 1.1 Schematic illustration of the human skin layer[4]. ... 2 Figure 1.2 Comparison of the number of publications related to research (Data collected from title search in Web of Science, Thomson Reuters on April 2021). ... 4
Figure 1.3 Single microneedles coated with different (A-G) types of drugs, (H-I) illustrate the microneedle patch and close up picture top view[18][19] ... 5
Figure 1.4 (a) cylinder silicon microneedles (bar=20 µm) [20], (b) a microfluid microneedle[21] ... 6
Figure 1.5 Schematic illustration of polymeric microneedle fabrication via PDMS micro-molding[25] ... 7
Figure 1.6 Drawing lithography fabrication process[30] ... 9 Figure 1.7 Schematic illustration of dissolving microneedle fabrication via droplet-born air blowing method [31]... 10
Figure 1.8 A stereolithographic 3D printer produces parts one layer at a time by photopolymerization[29]... 11
Figure 1.9 The 3D imprinted microneedle (A-F) The SEM images of the cone-shape microneedle, H) digital camera, and I) SEM images[15] ... 11
Figure 1.10 Schematic illustration (a-e) of the mechanism photopolymerization of self- focusing 3D lithography in PEGDA resin bath[32] ... 12 Figure 1.11 Design of the snake fang–inspired stamping patch. A) the venom delivery system of the rear-fanged snake B) Photograph of the snake fang–inspired MN array, C) photomasks with multi-blade patterns under the optimized UV exposure condition [35] ... 14
Figure 1.12 Simulation of the hydrodynamics of liquid drug delivery [35] ... 15 Figure 1.13 (a) Schematic illustration of mold-free fabrication set installation, (b) side view cross-section set up. ... 17
Figure 1.14 The schematic illustration of the concept of hydrogel drug delivery ... 18 Figure 1.15 Dextran structure ... 21 Figure 1.16 Sulfobetaine monomer... 22 Figure 2.1 A schematic of different microneedle drug release methods (a) hollow, (b) solid, (c) coated solid, (d) dissolving, and (e) hydrogel microneedles [1]. ... 33
Figure 2.2 (a) Injection of Rhodamine B solution into the agarose gel (b) microchannel entrance at the tip[2] ... 34
Figure 2.3 Solid microneedles fabricated out of (a-b) silicon, (c ) metal, and (d-f) polymer, imaged by scanning electron microscopy[4]. ... 35
Figure 2.4 Schematic illustration of the concept of dissolving microneedle, the encapsulated insulin was combined with starch/gelatin microneedles and dissolved rapidly in the skin to release encapsulated insulin[7]. ... 37
Figure 2.5 Schematic illustration of the double-layer drug coating microneedles. (a) the release profile of double-layer microneedle in agarose gel, (b) The merge of the optical and fluorescence image of double-layer drug release in real-time [6]. ... 38
Figure 2.6 A Schematic illustration of the hydrogel-microneedle drug delivery mechanism[10] ... 39
Figure 2.7 The digital images of maltose 200 mg/ml–MeHA microneedle patch before (i) and after removal (ii) (iii) zoom-in image of the microneedle tips after removing from mouse back[9]. ... 40
Figure 2.8 The chemical structure of AESO ... 41 Figure 2.9 The photomask fabrication process (a) sputtering methods (b) photomask drawing (c) photomask ... 43
Figure 2.10 microneedle fabrication set up ... 44 Figure 2.11 The photographs show the UV LED and power supply dimensions. ... 44
Figure 2.12 Schematic (a-d) illustration of the fabrication of microneedle arrays via inverted light UV lithography ... 46
Figure 2.13 The chemical structure of rhodamine B ... 47 Figure 2.14 Schematic illustration of the drug loading and rinsing method ... 48 Figure 2.15. Schematic illustration depicting the AESO sheet substrate fabrication. . 49 Figure 2.16. the flexible and transparent AESO sheets. ... 50 Figure 2.17 FTIR spectra of AESO sheet ... 51 Figure 2.18 (a) the methacrylate group on the substrate surface (b) the hydrogel binding with the surface substrate by covalent bonds[22] ... 51
Figure 2.19 1H-NMR spectra of the Dex-GMA for calculating the degree of substitution of GMA in D2O. ... 52
Figure 2.20. Photograph of the photomask pattern. ... 53 Figure 2.21 Light microscope images comparing the hypodermic needle with the microneedle different UV irradiation time fabrication (a) the hydrogel microneedle dye with rhodamine B (b) the dry microneedle dye with rhodamine B. ... 54
Figure 2.22 the microneedle on the fingertip comparing the size with a hypodermic needle ... 55
Figure 2.23 The schematic illustrates the UV light pathway in the microneedle fabrication and causing photopolymerization ... 56
Figure 2.24 photograph of the changing color of rhodamine B solution after 30 min loading in Dex-GMA/AAc microneedle patch. ... 57
Figure 2.25 The rhodamine B loading capacity; at t=0 min shows the dark pink color of the solution, and t=30 min shows the light pink color of the solution. ... 58
Figure 3.1 Poly-Sulfobetaine (poly-SPB) ... 65
Figure 3.2. Schematic illustration of the drug loading process in the microneedle patch.
... 67 Figure 3.3 Schematic illustration depicting fabrication of microneedle arrays via inverted light UV lithography. ... 71
Figure 3.4 Schematic illustration of the digital force gauge measuring setup. ... 72 Figure 3.5 Schematic illustration of the drug loading and sample collection process. 73 Figure 3.6 Schematic illustration depicting the drug release and sample collection process... 75
Figure 3.7 A) The rhodamine B loaded microneedles patch insertion onto porcine skin and covered with Tegaderm TM tap, B) the porcine skin after removing microneedles patch (After 30 min insertion). ... 78
Figure 3.8 FTIR spectra of cured Dex-GMA/SPB/AAc, Dex-GMA/SPB, and Dex- GMA/AAc... 80 Figure 3.9 EDX spectra of Dex-GMA/SPB/AAc microneedle. ... 80 Figure 3.10. A) The side-view image of rhodamine B loaded microneedle. B) The bird’s-eye view image of rhodamine B loaded microneedle. C) Microneedle and hypodermic needle with a fingertip for size comparison. D) SEM image. Photographs of parafilm after insertion of the four-point-star-shaped microneedle, E) the photograph captured during the light source project direct to the parafilm, and F) the light source project direct from behind the parafilm. ... 82
Figure 3.11 Swelling ratio of the hydrogel. Mechanical properties testing. ... 83 Figure 3.12 Force-displacement curve obtained from the compression testing of microneedles patches; the right-hand side shows the compression set-up. ... 85
Figure 3.13 Photograph of failure Dex-GMA/SPB/AAc microneedles presented the broken and shattered needles after compression. ... 85
Figure 3.14 A) Swelling ratio of the hydrogel. Mechanical properties testing B) Force-displacement curve obtained from the compression testing of microneedles patches; the right-hand side shows the compression set-up. C) The rhodamine B loading capacity; at t=10 min shows the dark pink color of the solution, and t=30 min shows the light pink color of the solution. D) Cumulative rhodamine B release over time. ... 88
Figure 3.15 The time lap of rhodamine B diffusion in 1% agarose. ... 89 Figure 3.16 Microneedle shape after rhodamine B solution uptake. The microneedle shape changed from pyramidal to lotus-like. ... 90
Figure 3.17. Time lap of the dextran-FITC diffusing from microneedle insertion into 1% agarose; the left-hand side shows the combination of fluorescent and bight field pictures, and the right-hand side shows only fluorescent pictures. ... 91
Figure 3.18 The penetration and diffusion testing of microneedle rhodamine B loaded on porcine skin. A) the picture shows the top-view, and B) the picture shows the side-view of cross-section; A) and B) were taken under white light; C) and D) were taken under UV light.
The yellow dot lines indicate where the needles pierced the porcine skin, and the pink color represents the rhodamine B diffusion in 20 min of the microneedle insertion before removal.
... 92 Figure 3.19 The protein loading and release at different pH values, A) extent of lysozyme loading percent at pH 7.4 to microneedles (bar graph) and the release to PBS (line graph), B) Insulin loading percent at pH 2 to microneedles (bar graph) and the release percent (line graph). ... 95
Figure 3.20 Change in absorbance at 340 nm due to reduction of NADH to NAD+ after being released from the polymers (shaded colors represent 95% confidence band) ... 98
Figure 3.21 The model of polymer structure loaded proteins (pH 7) of dry microneedle and hydrogel microneedle ... 98
Figure 3.22 Analysis of fibril formation of human insulin after release from polymers under heat at 45 °C for 3 days and without heat for 3 days determined by the ThT fluorescence assay. ... 99
Figure 3.23 Cytotoxicity of the extraction solution from different polymers. L929 cells were treated with a series of dilutions from the original concentration with 100, 50, 25, 12.5, and 6.25%. ... 100
Figure 3.24 Cytotoxicity of Dex-GMA and PEG-DA monomer from different polymers concentrations. L929 cells were treated with a series of concentrations. ... 100
1
Chapter 1
General Introduction
1.1 Transdermal drug delivery
For thousands of years, people have applied materials on the skin for treatment that was the basic therapeutic knowledge for the modern era of transdermal delivery. Recently, transdermal delivery has become a priority option for oral delivery and hypodermic injection.
Prausnitz et al. [1]categorized three generations of the transdermal delivery system from the first time introduced to the medical system. The first-generation transdermal delivery must have low molecular weight, lipophilic and low-dose drug. The second-generation transdermal delivery systems enhance the treatment by using different devices such as the iontophoresis machines and the non-cavitational ultrasound probes aim to control the rates of drug diffusion.
The third-generation delivery systems overcome the drug passing of stratum corneum layer barrier problems that allow delivering macromolecules, including therapeutic proteins and vaccines. The devices such as microneedles, thermal ablation, microdermabrasion, electroporation and cavitational ultrasound have been developed in this field. [1] The microneedle platform represents an attractive alternative to other methods; it is easy to self- administer and shows a high administered drug that reaches the systemic circul ation (bioavailability).[2]
2
1.1.1 Skin layer
The human skin is the largest organ in the human body which 2-3 mm thickness —the systemic circulation and outward flow of water, electrolytes and various substances. Human skin comprises two layers. An epithelium, called the epidermis, is the outermost layer, and the underneath layer, called the dermis, is connective tissue. The stratum corneum is located in the outermost layer of the epidermis and has about 10-30 μm. The stratum corneum behaves as the primary barrier between the body and the environment consists of about 20 layers of corneocytes. An extracellular matrix is located between the epidermis and dermis (Figure 1.1).
The dermis thickness varies from 500–2000 μm depending on the part of the body that lies below the epidermis. It is composed of fibroblasts, elastic fibers and collagens, filled in a ground substance composed of gelatinous proteoglycans and glycoproteins[3]. Extracellular fluid (ECF) means all body fluid outside the cells. The main component of the ECF is the interstitial fluid (ISF). For this situation, the microneedle for drug delivery can dissolve or extract the drug by the interstitial fluid below the stratum corneum and diffuse into the blood body circulation.
Figure 1.1 Schematic illustration of the human skin layer[4].
3
1.2 Microneedle
1.2.1 Microneedle technology
Microneedles (MNs) technology has been improving the efficiency of transdermal drug delivery. MNs can penetrate the stratum corneum layer but do not reach the nerve system[5][6]
and creates microchannels; hence the drug is administered directly into the epidermis or dermis layer, which enhances skin permeability, allowing the macromolecular drugs to enter into the systemic blood circulation.[7][8][9] In biomedical research, various materials are used to prepare MNs, including metals, silicon, and polymers, with the height of the needle ranging between 300-1,500 µm, and the base width between 100-500 µm[10][11] intended to delivery drug or diagnose. MNs have received widespread attention owing to their properties, such as painlessness, short time of skin trauma, possible self-administration, lack of bleeding, and high bioavailability.[12] In contrast, the other delivery methods have the risk of potential degradation associated with the gastrointestinal tract or first-pass liver effects.[13][14]
Recently, the properties and functions of microneedles have been receiving great attention from researchers worldwide, as seen by the increasing number of research articles in the field. Figure. 2a illustrates the increasing number of publications for overall microneedle research. In comparison, Figure 2b is emphasizing on the high output research on microneedle drug delivery only. The microneedle research areas include; sensors, medical devices, tissue adhesion[15], and diagnostic[16][17].
4 Figure 1.2 Comparison of the number of publications related to research (Data collected from title search in Web of Science, Thomson Reuters on April 2021).
1.2.2 Microneedle materials 1.2.2.1 Metal microneedle
The metal microneedles were fabricated by laser cutting process on the thin stainless steel sheets[18][19] and sterilized in a steam autoclave before use. The advantage of the metal microneedle is that its mechanical property is the strongest among all microneedle materials.
The metal microneedle patch (Figure 1.3) was inserted, with drug-coating, into the skin to create the microchannels to increase the skin permeability. In this case, the drug should rapidly dissolve from the needle because the metal microneedle should be removed as soon as possible to prevent the toxicity of the metal.
5 Figure 1.3 Single microneedles coated with different (A-G) types of drugs, (H-I)
illustrate the microneedle patch and close up picture top view[18][19]
1.2.2.2 Silicon microneedle
The silicon microneedle has a similar function as the metal microneedle, but the silicon microneedle process requires a cleanroom for fabrication. The silicon wafer was modified by a deep reactive ion etching process, consisting of anisotropic and anisotropic dry etching or dry etching, which depends on the design. Figure 1.4a illustrates that this fabrication technique creates the small-size cylinder shape microneedle array, and figure 1.4b shows the microfluid microneedle (hollow) device that can bypass the drug epidermis layer. However, silicon or ceramic microneedle has lower toxicity than metal microneedle, but it requires a clean room, an expensive, time-consuming process.
6 Figure 1.4 (a) cylinder silicon microneedles (bar=20 µm) [20], (b) a microfluid
microneedle[21]
1.2.2.3 Polymeric microneedle
The polymeric microneedle has gained more attention than other materials mentioned above due to its characteristics; (1) biocompatible, devoid of the immune response, (2) strong enough to penetrate stratum corneum layer, (3) controllable drug release, and (4) protecting drug damage or denature during the fabrication process (5) easy to fabricate. A wide range of polymer performance has been used for the structure of microneedle or drug cargo. The function of polymeric microneedle delivery can be categorized into three groups; dissolvable, bio-degradable, and swellable. For drug delivery, dissolvable and degradable have been more often developed because the interstitial fluid will dissolve and extract to the blood circulation.
In comparison, the swelling microneedle has developed to extract the interstitial fluid for diagnostic and delivery in the same microneedle.
7
1.3 Fabrication methods
There are two main strategies of MNs fabrication: mold-based fabrication and mold- less fabrication. The mold-based fabrication gives the exact shape of microneedle and shielding drug cargos. Some limitations of this technique are time-consuming, inflexible to change the MNs structure, need for extra pieces of equipment, and unsuitable for mass production. The most common mold-based process that has been used involves polydimethylsiloxane (PDMS) casting for micro-molding[22][23][24][25]. Briefly, starting with fabricating a female mold from the master template by pouring PDMS to the master microneedle, the PDMS female mold was peeled carefully once after PDMS solidifies. Micro-molds are filled with polymer (ingredient ) solutions using vacuum and centrifugal force to allow all cavities to fill with the solution. The polymer solution is solidified by air drying or UV irradiation, followed by removing the microneedle array from the mold (Figure 1.5). This method has only academic significance as it can be applied in only small-scale preparation [23][26], because it is time- consuming and requires a sterilization room to process.
Figure 1.5 Schematic illustration of polymeric microneedle fabrication via PDMS micro-molding[25]
8 On the other hand, mold-less fabrication such as drawing lithography, photolithography, and droplet-borne air blowing are suitable for the various structure of microneedle and allows tenability of height, base-width, and shape[17][27]. The 3D printing microneedle [28] allows the fabrication of complicated structures but is unsuitable for large- scale production due to the expensive materials required, time-consuming, and the requirement of a high-resolution 3D printer.[15][29]
Drawing lithography
The drawing lithography method is suitable for polymers which have high viscosity.
The process is performed in the range of melting temperature (Tm) to the glass transition temperature (Tg), such that during cooling down Tg the amorphous portion of polymer slowly change from the glassy liquid into the solid-state since the rearrangement occurs. In the fabrication process, the 2 identical circular plates are placed on the glassy liquid polymer (Figure 6). The polymer then adheres to the surface of circular plates; the glassy liquid gets elongated while moving apart of circular plates on decreasing temperature. The microneedle becomes narrow and solidifies around g before isolating between the upper and the lower plate.
Although this method can create an ultrahigh-aspect ratio microneedle, however, it does not provide microneedles with accurate size, requires a stable condition to fabricate, and only cone-shape microneedle fabrication is possible.
9 Figure 1.6 Drawing lithography fabrication process[30]
Droplet-borne air blowing
The droplet-borne air blowing process also uses two plates positioned face-to-face and then moving apart like the downing lithography, but it is applied air blowing to polymer droplets for solidification without heat or UV irradiation. In Figure 1.7, the polymer solution was dispensed, dropping for the base structure and following the drug-loaded polymer on the top. The top plate, which has the same dispensing drops were moved to contact the lower plate.
The plates were pulled apart gradually while the air was blowing. The liquid polymer became solid in a couple of mins, and the microneedle array was separated. The droplet-borne air blowing was commonly applied for the dissolvable microneedle compatible with chemical drugs and proteins, but it requires a specific machine for dropping polymer and sterilization room to process.
10 Figure 1.7 Schematic illustration of dissolving microneedle fabrication via droplet-
born air blowing method [31]
The 3D printing microneedle
Recently, 3D printers have provided a high resolution and have been developed for different purposes. It has been widely used for functional materials and microstructure fabrication. The researchers have designed different patterns and methods to fabricate microneedles to meet the specific functions. The 3D resin printers have been synthesized for many purposes, and many of them have been approved by the Food and Drug Administration, USA (FDA). Figure 1.8 illustrates the process of microneedle fabrication for the basic design [29]. The UV projector has generated the image projected to the mirror and reflected the resin bath to solidify microneedle layer to layer by photopolymerization. Figure 1.9 (A-F) shows the basic cone-shaped microneedle in different aspect ratios fabricated for the low-cost printer [29].
In contrast, figure (H-I) illustrates sophisticated microneedles printed for a high-resolution 3D printer for enhanced tissue Adhesion[15].
11 Figure 1.8 A stereolithographic 3D printer produces parts one layer at a time by
photopolymerization[29]
Figure 1.9 The 3D imprinted microneedle (A-F) The SEM images of the cone-shape microneedle, H) digital camera, and I) SEM images[15]
12 Photolithography
The photolithography microneedle fabrication overcomes the limitations of 3D printing; this method reduces the time consumed and the need for expensive instruments.
Photolithography using photomasks was used to fabricate polymeric microneedles in a single step[32]. The ultraviolet (UV) light dose was developed to batch presented a photoinitiator to form crosslinked polymeric 3D hydrogel microneedle structures (figure 1.10). During UV irradiation, the photoinitiator generates the free radicals that propagate the rapid polymerization reaction UV, and the light refracts at the noncured and photocured hydrogel (figure 10).
Therefore, the 3D microneedle shapes will depend on high-intensity light propagation.
Therefore, a short fabrication offers greater suitability of scaling up commercially for industrial purposes[33]. Some research using inclined/rotated UV lithography[33] or micro lensed UV lithography[34] was also applied to this method to enhance the UV light beam and intensity.
The inclined/rotated and micro-lensed methods were created and only a circular cone, whereas the multi-blade photomasks patterns can fabricate microneedle with the wings shape.
Figure 1.10 Schematic illustration (a-e) of the mechanism photopolymerization of self-focusing 3D lithography in PEGDA resin bath[32]
13 Most research used a polyethylene glycol diacrylate (PEGDA) [33] [32] [35] as a polymer resin that can produce the solid microneedles with a simple cone-shape or complicated 3D snake teeth (snake fang) [35] features without inclined/rotated and micro-lensed UV exposure, which the mechanical properties of the microneedles were depended on polymer- based resin.
The snake fang microneedle (Figure 1.11) is the most complicated microneedle shape classified as the solid microneedle. The 3D structure microneedle design depends on multi- blade patterns (Figure 1.11C). The increasing number of blade patterns will increase the number of microchannels on the side of the microneedle slope. Figure 1.12 shows the simulation of hydrodynamics of liquid drug delivery that the drug solution will supply from the PDMS drug reservoir.
However, this device shows several advances in the fabrication process and delivery system, but this fabrication process is time-consuming and many steps fabrication; therefore, it is not suitable for scaling up commercially for industrial applications.
14 Figure 1.11 Design of the snake fang–inspired stamping patch. A) the venom delivery system of the rear-fanged snake B) Photograph of the snake fang–inspired MN array, C)
photomasks with multi-blade patterns under the optimized UV exposure condition [35]
15 Figure 1.12 Simulation of the hydrodynamics of liquid drug delivery [35]
16 Herein, we developed the photolithography (mold-less ) technique; a micro-windows mask was prepared from a thin aluminum film on a glass slide and create the pattern by evaporating the aluminum layer with a high-resolution low-energy laser. UV irradiates to the micro-photomask perpendicularly to the polymer resin in the bath container, creates the conical path, and develops the 3D microneedle structure. In this study, we also developed the biocompatible photo-crosslinkable polymer from the dextran base polymer.
Our study developed the low-cost microneedle set up using only one UV LED (380- 400) nm as a light source and projected UV light upward that different from other research (Figure 1.13).
In addition, we synthesized the Dex-GMA/SPB/AAc, biocompatible polymer resin, that be able to cure by UV 380-400 nm. The cured polymer (dry) can swell water or biological liquid into the microneedle and become a hydrogel. This microneedle has many outstanding properties that overcome other methods and materials; 1) we can choose any drug for loading into the microneedle before using a cancer drug, insulin, antibody, antigen vaccine. 2) we can vary the dosage by adjusting the concentration of the original drug while loading; thus, we can minimize the drug usage and drug denaturation. 3) It is possible to process mass-scale production due to the short time fabrication; in this study, the fabrication and washing process is less than 5 min for a single microneedle patch and the low price of fabrication set up.
Figure 1.14 illustrates a concept of the novel microneedle for drug delivery. Figure 1.14a shows the drug is tapped in the polymer network of the dry microneedle. The drying state microneedle is strong enough to penetrate the stratum corneum skin layer and absorb the liquid from the epidermis layer. The swollen microneedle (Figure 1.14b) balances the drug concentration in hydrogel and out by releasing the drug to the reservoir. After completing delivery, the microneedle patch will be removed move out of the skin.
17 Figure 1.13 (a) Schematic illustration of mold-free fabrication set installation, (b) side
view cross-section set up.
18 Figure 1.14 The schematic illustration of the concept of hydrogel drug delivery
19
1.4 Drug delivery
Diabetes
The number of diabetes patients has increased from 108 million in 1980 to about 400 million in 2014[36]. Diabetes prevalence has been rising more rapidly in middle and low- income countries. The disease is a major cause of kidney failure, heart attacks, stroke blindness, and lower limb amputation. World Health Organization (WHO) reported that in 2016, 3.7 million deaths due to diabetes and high blood glucose and 1.5 million deaths were caused by diabetes. People with diabetes must inexhaustibly monitor their blood glucose levels every day and take the correct dose of the hormone insulin to maintain the normal blood glucose level.
The controllable insulin delivery using microneedle pad has introduced a new way of diabetes treatment to overcome painfulness and an imprecise insulin dosage injection.
Comparing to oral and nasal drug delivery, the drug delivery across the skin also avoided the hepatic first-pass extraction and delivered to the blood circulation at a pharmacologically relevant rate.
20
1.5 Hydrogel
1.5.1 Hydrogel in drug delivery
Hydrogel is a three-dimensional polymer network that occurs by the crosslinking of polymer chains via physical interaction, primary covalent crosslinks, hydrogen bonding, polymer crystallites, and Van der Waals interaction. The high-water content of hydrogels has received significant attention because of their related potential in t pharmaceutical industries.
The hydrogel has a high (70-99%) absorption of water and biological fluid due to its functional group such as -OH, CONH2, -SO3H, -CONH, and -COOR. The swollen microneedles are soft and rubbery surface structures and physicochemical properties similar to human tissue, so it will feel pain during inserting into the skin while drug release. The hydrogel microneedle has excellent biocompatibility and the capability to load drug and release into the skin. These characteristic features make hydrogels a potential candidate for microneedle drug delivery.
Hydrogel applications for controlled drug delivery systems (DDS) have become very popular in recent years. In this study, the potentiality of hydrogel for microneedle drug delivery has been explored to reduce drug release rate compared with dissolving microneedle. In previous research, our hydrogel: Dex-GMA/AAc base hydrogel performed the charge interaction with positive charge drug and proteins; consequently, the drug can be entrapped in this microneedle hydrogel.
21
1.5.2 Dextran
Dextran is a polysaccharide synthesized by fermentation of Leuconostoc mesenteroids bacteria when grown on sucrose-containing media, consisting of α-1,6 linked glucopyranoside a small percentage of α-1,3 linked residues (Figure 1.15). Dextran fractions can vary from 1kDa to 2,000kDa molecular weight that contains a large amount of hydroxyl group leading to high hydrophilic properties and capability for chemical modifying. Dextran has been selected for biomedical applications because of its low toxicity[37] and biocompatibility[12][20]. Dextran can degrade and is removed from the human body by various parts such as the colon, spleen, and liver.
Figure 1.15 Dextran structure
22 Zwitterionic polymers
The Zwitterionic polymer is a sub-category of a polyampholytes group with unique characteristics[31] consisting of the anionic and cationic groups, commonly on the same repeating unit. The ionic groups are functional over a large pH window. Therefore, the total charge of the zwitterionic polymer in the normal state is equal to zero; polyzwitterions overall charge neutrality exhibit a different, hybrid-like property profile. Furthermore, the zwitterionic polymer reveals a high hydrophilic property [38]. The zwitterionic polymer has similar properties to proteins[39]. The polysulfobetaines (poly-SPB) show the biocompatible properties that resemble phospholipids in recent research[40][41][42]. Sulfobetaine polymers show antifouling properties[43], suppress the nonspecific binding of proteins and do not modify the structure of the hydrogen-bonded network of water molecules at the interface of the polymer and material[41]. In the drug delivery system research, sulfobetaine polymers have been developed due to the polymer exhibited high efficiency inhibits protein aggregation[44][45][46].
In this study, SPB (Figure 1.16) was combined with the Dex-GMA/AAc hydrogel network to prevent protein aggregation during loading proteins into the microneedle patch. The proteins are attached to the SPB side chains in the polymer structure to inhibit protein aggregation or prevent protein denature.
Figure 1.16 Sulfobetaine monomer
23
1.6 Smart microneedle
This study combined the zwitterionic polymers (SPB) to the main polymer structure (Dex-GMA) to suppress protein aggregation and protein denaturation. In the field of biopharmaceutics, protein instability[25][26][27] is an ongoing challenge for transdermal drug delivery, especially microneedle technology. Protein denaturation and aggregation are some of the primary causes of protein instability[27]. The uniqueness of zwitterion polymers is fascinating in this regard. In our previous report, zwitterionic polymer systems showed significant efficiency against the thermal aggregation of lysozymes and insulin at very low polymer concentration and over 24h incubation periods[27][28][29][30].
In this study, we prepared a photo-crosslinkable hydrogel by introducing glycidyl methacrylate (GMA) through dextran chains to create the double bond modified dextran (Dex- GMA)[21][22][47]. To create a smart microneedle platform, especially for protein drug delivery. Then the Dex-GMA was copolymerized with Sulfobetaine (SPB) and acrylic acid (AAc) to give the Dex-GMA/SPB/AAc hydrogel. PAA has high water uptake capability (super absorbent) due to the very high swelling ratio[48]. PAA hydrogels have been widely used in drug delivery patches by their bioadhesive property due to the hydrogen bonding and covalent bonding ability of such materials. In a recent study, Dex-GMA/PAA microgel particles were applied for a wound dressing as a hemostatic agent[49]. PAA also has anionic properties due to the carboxyl group releasing the drugs by electrode pad.
Herein, we report a novel microneedle fabrication method from biocompatible materials and increased drug delivery performance by combining it with protein inhibition polymer. This mold-free fabrication technique is suitable for the mass production of swelling microneedle at a low price. We developed Dex-GMA/SPB/AAc for the first time for photolithography microneedle fabrication which took less than 1 min for photopolymerization for one microneedle patch. The microneedle substrate is made from acrylated epoxidized
24 soybean oil (AESO) that does not absorb water while loading the drug, and all mixed solutions will swell into the hydrogel MNs only. Furthermore, the hydrogel microneedle patch was loaded with protein drug and the modified zwitterion polymer side chain for protein pharmaceutical aggregation inhibition. In this report, the fabrication process, morphology, rhodamine B diffusion, and drug release performance are investigated.
1.7 Objective of study
Microneedle technology has attracted increasing interest in medical research. However, the recent microneedle fabrication has some drawbacks; for example, almost all the microneedle is fabricated by mold required the vacuum and centrifugal system, time- consuming, and unsuitable for massive production. The mold-less fabrication techniques require expensive equipment and a special room for fabrication. Even though 3D printing gives the precision microneedle, it has a limitation of material of resin, time-consuming, and the high cost of the printer so, it is not suitable on an industrial scale.
This study aims to develop the microneedle fabrication technique that can produce on a mass scale using the polymeric materials that our group has been developed [50] [44][46]. In this study, the Dex-GMA [50] and inhibitor polymer for protein aggregation[44][46] have been synthesized for photolithography microneedle fabrication. The unique properties of novel microneedles create tremendous opportunities for various potential drug loading and release;
the microneedle patch may serve as minimal transdermal drug delivery such as Alzheimer’s disease, cancer treatment, diabetes, and cell therapy.
25 The research objectives proposal are
1. To synthesize the curable biocompatible polymer resin that inhibits protein aggregation and has swelling and releasing properties.
2. To develop the photolithography (mold-less) fabrication technique by using the curable biocompatible polymer resin.
3. To study the capacities of chemical and protein loading and drug loading to microneedle patches.
4. To study drug diffusion properties of microneedle on the artificial skin.
5. To study the protein function after releasing from microneedle patch.
26
1.8 References
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33
Chapter 2: Polymer synthesis and mold-less microneedle fabrication
2.1 Introduction
Over the past decades, microneedle drug delivery systems have attracted increasing attention in biomedical research. It offers the advantages of new active pharmaceutical ingredients (API) for the following reasons; (1) the large molecules can be administered, (2) enhanced drug efficiency in the reduction dosage, (3) targeted to the skin area to delivery, (4) faster healing after administration. The drug delivery microneedles (Figure 2.1) are classified into 5 types according to their functions: hollow, solid, coated solid, dissolving, and hydrogel microneedles[1].
Figure 2.1 A schematic of different microneedle drug release methods (a) hollow, (b) solid, (c) coated solid, (d) dissolving, and (e) hydrogel microneedles [1].
34
2.1.1 Microneedle Classifications
Hollow microneedle can be used for infusion liquid formulation flows (typically, 10- 100 μL/min) from the reservoir into the skin through the hollow microneedles. Hollow microneedle can deliver a higher API than other microneedle delivery methods. The biggest drawback found in this method is a clogging problem in the needle tip in the tissue and requires a microfluidic chip or micropump connected to the microneedle array. Figure 2.2a shows an in-plane single-crystal-silicon microneedle array that releases rhodamine B into the agarose gel with about 20 µm diameter microchannel (Figure 2.b).[2]
Figure 2.2 (a) Injection of Rhodamine B solution into the agarose gel (b) microchannel entrance at the tip[2]
35 Solid microneedles are commonly used for skin pre-treatment in the field of cosmetic medicine[3]. The microneedles pierce the skin to create the microchannels in the stratum corneum layer, the conventional drug in the form of dream, solution, or gel is applied in this region. Solid microneedle can make from metal, ceramic and polymer (Figure 2.3).[4] The solid microneedles can fabricate by the etching process for silicon or metal and micro-molding for polymer.
Figure 2.3 Solid microneedles fabricated out of (a-b) silicon, (c ) metal, and (d-f) polymer, imaged by scanning electron microscopy[4].
Coated solid microneedles (coat and poke approach) are the solid microneedle coated with a water-soluble drug formulation. After inserting microneedle, the coated drug dissolves and then continuously releases into the skin. Although the solid microneedle allows rapid
36 therapeutic pharmaceutical ingredients (APIs) delivery, less than 0.7 µg/needle of materials can be coated.[5]
Dissolving microneedles (poke and release) consist of biodegradable polymers or sugars-based matrices that can be completely dissolved or degraded in the skin. This method is easy to fabricate by micro-molding and has increased attention recently. The delivery rate is controlled by adjusting the polymeric composition of the microneedle array in the fabrication process. The microneedle may be used device for applying to the skin. The interstitial fluid under the skin dissolves the microneedle material-based rapidly, and the drug has released into the skin and diffuses to the blood vessel over time.
Recently, dissolvable water and degradable material have been used for microneedle fabrication, such as sugars, galactose, dextran, pullulan[5], gelatin[6], sodium hyaluronate (HA)[7], poly(vinylpyrrolidone), carboxymethyl cellulose (CMC), hydroxy-propyl-methyl- cellulose (HPMC)[8], chitosan[7], chitin, silk[9], and poly(vinylpyrrolidone)[10]. Figure 2.4 illustrates the concept of dissolving microneedle; the drug was dissolved in the water-soluble or degradable material(s). The mixed solution was poured into the micro-molding followed by centrifugal or vacuum to force the liquid to fill all gaps in the mold. The microneedle was taken off 6-24 h after drying in the mold.
The advantages of this method are the low cost of polymeric materials and processes at the ambient temperature that is possible for industrial mass production. The disadvantages are that the process requires a vacuum system or centrifugal methods to fill the liquid into the micro-mold, which is time-consuming.
The dissolving microneedle is also used for ocular drug delivery for eye diseases and injuries[6]. Aung Than et al. [6] enhanced therapeutic efficacy by the double-layered micro- reservoirs by using corneal neovascularization as the disease model. One eye microneedle patch can produce about 90% reduction of the neovascular area by delivering an anti-
37 angiogenic monoclonal antibody (DC101). They proposed that the eye patch microneedle for intraocular drug delivery method possibly to self-treatment at home for many eye diseases.
Figure 2.5 shows the diffusion of two fluorescence drugs diffusion, IgG(680) (red) dissolving in HeMA, IgG(488)(green) dissolving in HA, in agarose gel. The outer layer (IgG 488) was rapidly diffused to the agarose gel first, then after 30 min, the inner drug (IgG 680) was diffused throughout the gel later.
Figure 2.4 Schematic illustration of the concept of dissolving microneedle, the encapsulated insulin was combined with starch/gelatin microneedles and dissolved rapidly in
the skin to release encapsulated insulin[7].
38 Figure 2.5 Schematic illustration of the double-layer drug coating microneedles. (a) the release profile of double-layer microneedle in agarose gel, (b) The merge of the optical
and fluorescence image of double-layer drug release in real-time [6].
39 Hydrogel (swellable) microneedles consist of an insoluble polymeric network. The dry polymeric microneedle is inserted into the skin. The microneedles are uptaken the interstitial fluid around the tissue and become a hydrogel. The polymer network has swelled the liquid and extended the size of the pore so that it allowed the drug to diffuse from a higher concentration (hydrogel) to a lower concentration (tissue). Figure 2.6 illustrates the mechanism of drug release via hydrogel microneedle
The hydrogel microneedles have two functions to overcome the limitation of dissolving microneedle: (1) absence of microneedle material in the skin after removing the microneedle patch; (2) the treatment able to terminate if adverse drug reactions or overdose occur. The crosslinking density of the polymer controls the swelling and releasing properties, but it has to maintain the microneedle strength to penetrate the stratum corneum and epidermic layer.
Before removing the microneedle, the swelled hydrogel should retain the mechanical toughness to remove all materials from the skin[8]. Figure 2.7 shows that the microneedle retains the swollen microneedle structure after being removed from mouse back skin[9].
Figure 2.6 A Schematic illustration of the hydrogel-microneedle drug delivery mechanism[10]
40 Figure 2.7 The digital images of maltose 200 mg/ml–MeHA microneedle patch before (i) and after removal (ii) (iii) zoom-in image of the microneedle tips after removing from
mouse back[9].
The microneedle drug delivery methods have provided a versatile platform to increase transdermal drug delivery efficiency. Currently, the microneedle technology applications have been developed to deliver from a common drug; vitamins, minerals, ibuprofen, and advanced drug treatment such as donepezil hydrochloride (DPH) used for the treatment of Alzheimer's disease[11], anticancer drugs; doxorubicin (DOX)[12][13], and trametinib (Tra)[13], antibody, antigen[14], insulin[7], lysozyme[15], vaccine[16][17][18], and cells for cell therapy[19].
Generally, the researchers have combined the drug with microneedle materials solution before the microneedle fabrication process. So, there are some drawbacks to those methods: (1) the drug denaturation before use, (2) fixed drug dosages per microneedle patch, (3) impossible to deliver on-demand purpose. In this study, a new type of microneedle patch was designed to overcome those limitations; the dry microneedle can load several types of drugs all on-demand, for example, cancer drug treatment, insulin, the vaccine that can adjust dose before loading to microneedle patch.
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2.2 Materials and Methods 2.2.1 Materials
Dextran (molecular weight (MW) = 70 kDa) was obtained from Meito Sangyo (Nagoya, Japan). Glycidyl methacrylate (GMA) and 4-dimethyl aminopyridine (DMAP), Acrylic acid (AAc), ethylene glycol dimethacrylate (EGDMA), 2,2-Dimethoxy-2- phenylacetophenone (DMPA), acrylated epoxidized Soybean oil (AESO) (Figure 2.8), and rhodamine B were obtained from Sigma-Aldrich(St. Louis, MO, USA). 2,2'-Azobis(2- methylpropionamidine) dihydrochloride was obtained from Wako Pure Chemical Industries (Osaka, Japan). EGDMA and AAc were used with purification.
2.2.2 Synthesis of AESO resin
The AESO resin was synthesized for making a substrate of the microneedle array. First, the photoinitiator, DMPA, 2g, was dissolved in 20 ml of EGDMA. The mixed solution was added to the 180 ml AESO in the round brown bottle and stirred for 24 h in a dark room at room temperature. The AESO resin was covered with aluminum foil and stored at room temperature. FTIR analysis was conducted using FT-IR spectrometer spectrum 100 PerkinElmer in 4000-380 cm-1 to investigate the functional groups of synthesized products.
Figure 2.8 The chemical structure of AESO
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2.2.3 Synthesis of Dex-GMA
Dextran-glycidyl methacrylate (Dex-GMA) synthesis was synthesized using our previously reported method.[20] In brief, 20 g of dextran was slowly added to 80 mL of dimethyl sulfoxide (DMSO), and the solution was stirred until complete dissolution. The transparent solution was then stirred for another 30 min under nitrogen gas. Then, 3.2 g of DMAP and 8.8 g of GMA were added to the solution and purged nitrogen gas for 30 min. The mixture was stirred at 50 °C overnight before neutralization by 6.5 mmol hydrochloric acids (HCl). The mixture was dialyzed against distilled water for 7 days using a dialysis membrane (MWCO = 3.5 kDa). The resulting product was dried in the oven for 48 h at 47 °C, followed by vacuum drying for 48 h at 25 °C to obtain Dex-GMA derivative as a pale yellow-brown flake product.
The synthesized material was characterized by 1H nuclear magnetic resonance (NMR) (400 MHz equipped with a cryogenic probe, Bruker). The results of the 1H-NMR spectroscopy were used to investigate the degree of substitution (%DS).
2.2.4 Synthesis of Dex-GMA/AAc
The hydrogel resin was prepared by 2g of Dex-GMA was dissolved in 3.5ml distilled water at 50 °C in a brown glass tube and stirred until they were completely dissolved. The photoinitiator of 2,2'-Azobis(2-methylpropionamidine) dihydrochloride, 125 mg, was added to the mixed solution. Then, 4 ml of acrylic acid and DMPA, the photoinitiator, 150 mg, were added to the mixed solution and stirred for 2 h at room temperature. The resin was covered with aluminum foil and stored in the fridge (4-5 °C) for long-term use.
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2.2.5 Mold-less Microneedle Fabrication 2.2.5.1 Photomask
The photolithography method was used for the microneedle fabrication arrays. A photomask was used for blocking UV light, preventing photopolymerization outside of microneedle fabrication areas. Briefly, a glass slide was coated by a single-side aluminum 40 nm thick by a sputtering technique (Figure 2.9a). Next, the low-energy laser was used for evaporating the thin aluminum layer to create the four-points star micro-windows mask array (Figure 2.9b) with a based-diameter 400 μm (Figure 2.9c). The micro-size patterns were aligned in a 6 mm x 6 mm square area. The UV light passing photomask was generated the free radical in the polymer resin in the fabrication process, causing a photopolymerization and creating 3D microneedle structure patterns.
Figure 2.9 The photomask fabrication process (a) sputtering methods (b) photomask drawing (c) photomask
